Bottom Line:
The Ca(V)beta/Ca(V)alpha(1)-I-II loop and Ca(2+)/calmodulin (CaM)/Ca(V)alpha(1)-C-terminal tail complexes have been shown to modulate each, respectively.Nevertheless, how each complex couples to the pore and whether each affects inactivation independently have remained unresolved.Collectively, the data strongly suggest that components traditionally associated solely with VDI, Ca(V)beta and the IS6-AID linker, are essential for calcium-dependent modulation, and that both Ca(V)beta-dependent and CaM-dependent components couple to the pore by a common mechanism requiring Ca(V)beta and an intact IS6-AID linker.

Affiliation: Cardiovascular Research Institute, Department of Biochemistry and Biophysics, California Institute for Quantitative Biosciences, University of California, San Francisco, CA 94158, USA.

ABSTRACTTwo processes dominate voltage-gated calcium channel (Ca(V)) inactivation: voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). The Ca(V)beta/Ca(V)alpha(1)-I-II loop and Ca(2+)/calmodulin (CaM)/Ca(V)alpha(1)-C-terminal tail complexes have been shown to modulate each, respectively. Nevertheless, how each complex couples to the pore and whether each affects inactivation independently have remained unresolved. Here, we demonstrate that the IS6-alpha-interaction domain (AID) linker provides a rigid connection between the pore and Ca(V)beta/I-II loop complex by showing that IS6-AID linker polyglycine mutations accelerate Ca(V)1.2 (L-type) and Ca(V)2.1 (P/Q-type) VDI. Remarkably, mutations that either break the rigid IS6-AID linker connection or disrupt Ca(V)beta/I-II association sharply decelerate CDI and reduce a second Ca(2+)/CaM/Ca(V)alpha(1)-C-terminal-mediated process known as calcium-dependent facilitation. Collectively, the data strongly suggest that components traditionally associated solely with VDI, Ca(V)beta and the IS6-AID linker, are essential for calcium-dependent modulation, and that both Ca(V)beta-dependent and CaM-dependent components couple to the pore by a common mechanism requiring Ca(V)beta and an intact IS6-AID linker.

fig5: Effects of CaVβ isoforms on calcium inactivation stem from underlying effects on VDI. Normalized inactivation curves measured at +20 mV for (A) CaV1.2 and (B) CaV1.2 GGG subunits coexpressed with CaVβ1, CaVβ2a, CaVβ2b, or in the absence of CaVβ. (Left) VDI is shown and is reproduced for comparison from the first 300 ms of Fig. 3 (A and B). (Middle) Inactivation in calcium. (Right) netCDI.

Mentions:
In light of the clear rank order effects that different CaVβ isoforms have on VDI, we asked whether there were similar differences in netCDI when different CaVβs were present. Wild-type CaV1.2 channels coexpressed with CaVβ1, CaVβ2a, or CaVβ2b show small differences in CDI but no real difference when netCDI is considered (τ values of 20–30 ms) (Fig. 5 A and Table I). Incorporation of the triple-glycine mutant in the CaV1.2 IS6-AID linker causes a reduction in CDI in the presence of CaVβ1, CaVβ2b, and CaVβ2a (τ1 = 200–350 ms; Table I). Even though there appear to be differences between the CaVβ isoforms with respect to inactivation in calcium (Fig. 5 B, middle, and Table S1), these distinctions are due almost exclusively to the underlying VDI differences (Fig. 5 B, left) and vanish when one considers netCDI (Fig. 5 B, right, and Table I). Together with the HotA CDI results, these data suggest that although a CaVβ subunit is required for CDI, all CaVβ isoforms support netCDI equally. Thus, the primary route for coupling CDI components to the pore would appear to be through the IS6-AID linker and not through secondary interactions sites that contribute to isoform-specific VDI modulation differences.

fig5: Effects of CaVβ isoforms on calcium inactivation stem from underlying effects on VDI. Normalized inactivation curves measured at +20 mV for (A) CaV1.2 and (B) CaV1.2 GGG subunits coexpressed with CaVβ1, CaVβ2a, CaVβ2b, or in the absence of CaVβ. (Left) VDI is shown and is reproduced for comparison from the first 300 ms of Fig. 3 (A and B). (Middle) Inactivation in calcium. (Right) netCDI.

Mentions:
In light of the clear rank order effects that different CaVβ isoforms have on VDI, we asked whether there were similar differences in netCDI when different CaVβs were present. Wild-type CaV1.2 channels coexpressed with CaVβ1, CaVβ2a, or CaVβ2b show small differences in CDI but no real difference when netCDI is considered (τ values of 20–30 ms) (Fig. 5 A and Table I). Incorporation of the triple-glycine mutant in the CaV1.2 IS6-AID linker causes a reduction in CDI in the presence of CaVβ1, CaVβ2b, and CaVβ2a (τ1 = 200–350 ms; Table I). Even though there appear to be differences between the CaVβ isoforms with respect to inactivation in calcium (Fig. 5 B, middle, and Table S1), these distinctions are due almost exclusively to the underlying VDI differences (Fig. 5 B, left) and vanish when one considers netCDI (Fig. 5 B, right, and Table I). Together with the HotA CDI results, these data suggest that although a CaVβ subunit is required for CDI, all CaVβ isoforms support netCDI equally. Thus, the primary route for coupling CDI components to the pore would appear to be through the IS6-AID linker and not through secondary interactions sites that contribute to isoform-specific VDI modulation differences.

Bottom Line:
The Ca(V)beta/Ca(V)alpha(1)-I-II loop and Ca(2+)/calmodulin (CaM)/Ca(V)alpha(1)-C-terminal tail complexes have been shown to modulate each, respectively.Nevertheless, how each complex couples to the pore and whether each affects inactivation independently have remained unresolved.Collectively, the data strongly suggest that components traditionally associated solely with VDI, Ca(V)beta and the IS6-AID linker, are essential for calcium-dependent modulation, and that both Ca(V)beta-dependent and CaM-dependent components couple to the pore by a common mechanism requiring Ca(V)beta and an intact IS6-AID linker.

Affiliation:
Cardiovascular Research Institute, Department of Biochemistry and Biophysics, California Institute for Quantitative Biosciences, University of California, San Francisco, CA 94158, USA.

ABSTRACTTwo processes dominate voltage-gated calcium channel (Ca(V)) inactivation: voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). The Ca(V)beta/Ca(V)alpha(1)-I-II loop and Ca(2+)/calmodulin (CaM)/Ca(V)alpha(1)-C-terminal tail complexes have been shown to modulate each, respectively. Nevertheless, how each complex couples to the pore and whether each affects inactivation independently have remained unresolved. Here, we demonstrate that the IS6-alpha-interaction domain (AID) linker provides a rigid connection between the pore and Ca(V)beta/I-II loop complex by showing that IS6-AID linker polyglycine mutations accelerate Ca(V)1.2 (L-type) and Ca(V)2.1 (P/Q-type) VDI. Remarkably, mutations that either break the rigid IS6-AID linker connection or disrupt Ca(V)beta/I-II association sharply decelerate CDI and reduce a second Ca(2+)/CaM/Ca(V)alpha(1)-C-terminal-mediated process known as calcium-dependent facilitation. Collectively, the data strongly suggest that components traditionally associated solely with VDI, Ca(V)beta and the IS6-AID linker, are essential for calcium-dependent modulation, and that both Ca(V)beta-dependent and CaM-dependent components couple to the pore by a common mechanism requiring Ca(V)beta and an intact IS6-AID linker.